We will develop a human sickle cell mouse model for designing and testing gene therapy treatments of sickle cell disease and for functional studies of sickling in vivo. The sickle cell mouse line will be produced using a human beta globin locus yeast artificial chromosome containing the beta-S gene (beta-S-YAC) and a human alpha globin gene YAC (alpha-YAC) in a mouse homozygous for murine alpha and beta globin gene knockout mutations. This line will synthesize exclusively, or mostly, human hemoglobin S (Hb S) in the adult and human fetal globin (Hb F) and Hb S in the fetus. Hb F synthesis will rescue fetuses from lethal red cell sickling that would result from exclusive production of Hb S. We will extend the usefulness of this model by developing a novel approach for targeted mutagenesis transgenes directly fertilized oocytes derived from transgenic mice. The goal of the method is to provide an easy means to substitute , in vivo, the human alpha or beta-S genes with alleles encoding mutant alpha or beta-S chains that may inhibit or induce sickling. We will utilize the bacteriophage P1 Cre/lox and the yeast FLP/FRT site-specific recombination systems to catalyze gene replacement. Utilization of the sickle cell line, in conjunction with this targeted mutagenesis method, to study in vivo inhibition of sickling will have a major impact on designing vectors for gene therapy of sickle cell disease, as well as enhancing the ability of other investigators to study the pathophysiology of sickling. The sickle cell mouse model will also be employed for testing the expression of trans-acting factors that potentially reverse the developmentally- regulated fetal gamma globin to adult beta globin switch as an alternative form of sickle cell gene therapy.